CN1785425A - Preparation method of poly peptide vaccine for treating lefteye flounder adenolymphocele - Google Patents
Preparation method of poly peptide vaccine for treating lefteye flounder adenolymphocele Download PDFInfo
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Abstract
A process for preparing the polypeptide vaccine of the lymphocystis of bastard halibut includes such steps as analyzing the biologic information of the genomic sequence of the European strain of lymphocystis virus to obtain 3 simulative epitops of the nucleocapsidprotein and membrane protein for the relative Chinese strain, synthesizing 3 polypeptides according to their amino acid seguences, coupling with protein carrier, and purifying.
Description
Technical field
The present invention relates to the preparation method of the polypeptide vaccine of lefteye flonder lymphocystic disease.
Background technology
(Lymphocystis disease LCD) is puzzlement culture fishery one of insurmountable difficult problem for many years to the lymphocyst disease.Its pathogen, lymphocystis disease virus (Lymphocystis disease virus LCDV) can infect comprise Paralichthys olivaceus 100 surplus kind of sea water and fresh water Fish.With regard to Paralichthys olivaceus, the sick fish that infects LCDV produces a large amount of knobs at body surface, has a strong impact on its economic worth, should disease also can cause culturing the mortality rate of Paralichthys olivaceus up to 60-70% simultaneously.Because Paralichthys olivaceus is the main economic kind of China's sea-farming, and the lymphocyst disease has high epidemic characteristics, and is very huge to the economic loss that aquaculture causes.Therefore, pressing for effective medicine or vaccine is prevented and treated.
Vaccine has been proved to be one of the most effective diseases prevention and treatment means.Through constantly development, the subunit of the third generation (polypeptide) vaccine and the nucleic acid vaccine in the 4th generation are progressively substituting the emphasis that traditional deactivation and attenuated vaccine become the vaccine research and development.Subunit vaccine is applied in the mankind's hygiene and health system at present, comprises the control of viral diseases such as hepatitis B, influenza.The characteristics of subunit vaccine are that safety is good, can induce body CD4+T cell by plasmacytic differentiation of Th1 pathway activation and propagation effectively, thereby improve the humoral immunoresponse(HI) level.
The core of subunit vaccine development is that the subunit structure of antigen protein must keep original immunogenicity and immunoreactivity.The antigen protein quantity of known structure and function is very little, occupy the research and development progress that most unknown antigen albumen has seriously restricted subunit vaccine, this is that need expend a large amount of time and manpower and materials could solve because proteinic separation, purification and structural functional analysis remain a technical barrier in today that life sciences has obtained quantum jump.On the contrary, the develop rapidly of gene/genomic clone, sequencing technologies provides strong supplementary means for reverse protein function, structural analysis.Along with the nucleic acid database of magnanimity constantly increases with the geometrical progression form, bioinformatics contains the information strong tool and also arises at the historic moment as excavating sequence, and is in the high speed development process.
The present invention utilizes bioinformatics technique to infer the mimic epitope of generation, according to the polypeptide of the synthetic corresponding mimic epitope of their aminoacid sequence, through preparing the polypeptide vaccine of specificity at the lefteye flonder lymphocystic disease with the coupling of carrier.
Summary of the invention
The objective of the invention is to rely on three mimic epitopes of existing LCDV-cn protective antigen nucleocapsid protein and envelope protein, make up the mimic epitope polypeptide vaccine of specificity at the lefteye flonder lymphocystic disease.
The preparation method of the polypeptide vaccine of lefteye flonder lymphocystic disease of the present invention is, the bioinformatic analysis that utilization is carried out known lymphocystis disease virus Europe separated strain (LCDV-1) genome sequence and obtain 3 mimic epitopes of Paralichthys olivaceus LCDV-cn (lymphocystis disease virus Chinese pathogenic strain) nucleocapsid protein and envelope protein, then according to the aminoacid sequence of mimic epitope, synthesize and obtain corresponding 3 peptide species, through with the coupling and the purification of protein carrier, promptly prepared the mimic epitope polypeptide vaccine of lefteye flonder lymphocystic disease.And then, verified the specificity and the protectiveness of polypeptide vaccine by culturing the zoopery of Paralichthys olivaceus.
3 above-mentioned mimic epitope sequences are respectively (aminoacid in the literary composition is called for short the IUPAC standard that all adopts):
29A:TKNNMSRTSETDNN, i.e. NH
2-Thr-Lys-Asn-Asn-Met-Ser-Arg-Thr-Ser-Glu-Thr-Asp-Asn-Asn-COOH
29B:CKKNSDSTDC, i.e. NH
2-Cys-Lys-Lys-Asn-Ser-Asp-Ser-Thr-Asp-Cys-COOH
TK3:TNEERRLMGT, i.e. NH
2-Thr-Asn-Glu-Glu-Arg-Arg-Leu-Met-Gly-Thr-COOH
The protein carrier that adopts is a key hole maple hemocyanin (abbreviating KLH as), also can adopt hemoglobin, bSA etc.
The principle of institute of the present invention foundation is as follows: one, LCDV-1 and LCDV-cn belong to Iridoviridae, Lymphocystivirus together, have higher homology (homology of nucleocapsid protein Gene MCP on nucleotide sequence of known two kinds of viruses is 77%) on the sibship, therefore be suitable for carrying out comparison, the analysis of nucleotide sequence.Simultaneously; because the host of two kinds of virus infections and tissue, cell pathological reaction and symptom similar, that cause are also identical; the receptor response of two kinds of virus generations also is similar to the seroimmunity reaction, can infer in view of the above, has consistent protective antigen between two kinds of viruses.Determined in view of the genome sequence of LCDV-1, utilize the genome sequence of LCDV-1 to infer that therefore the protective antigen of LCDV-cn and antigenic determinant thereof become possibility.
Its two, antigenic determinant (or claim epi-position) is that protective antigen induces body to produce the key of adaptive immune response.The complex that MHC I, II molecule and epi-position form is respectively T
CAnd T
HDiscern, cause cytokine secretion, B, T cell activation, propagation and break up, finally produce corresponding body fluid and cellullar immunologic response.Therefore, utilizing epi-position directly to make up polypeptide vaccine can induce body to produce specific immune response.
The specific embodiment
The Paralichthys olivaceus LCDV-cn protective antigen mimic epitope that this method utilization obtains through bioinformatic analysis; synthetic obtaining and the corresponding to polypeptide of mimic epitope sequence; through with the coupling and the purification of carrier protein, prepare the mimic epitope polypeptide vaccine of lefteye flonder lymphocystic disease.Concrete grammar is as follows:
At first select Paralichthys olivaceus LCDV-cn protective antigen mimic epitope: by the bioinformatic analysis that LCDV-1 carried out to known group sequence; screening obtains 3 mimic epitopes of two kinds of protective antigen nucleocapsid proteins of LCDV-cn and envelope protein, i.e. TK3,29A and 29B.Simultaneously; in order to discern different angtigen presentation approach to the issuable influence of polypeptide vaccine; and the specificity of mimic epitope polypeptide vaccine, chosen totally three of the canonical sequence (TK1) of above-mentioned two kinds of protective antigen mimic epitopes and control sequence (TK2,29C) respectively.Choose mimic epitope and the explanation of reference and control sequence be listed in the table below:
| Protective antigen | The mimic epitope sequence | Length | Explanation |
| Nucleocapsid protein TK TK1 TK2 TK3 envelope protein ORF29 29A 29B 29C | AIKLKADN RMNNNGTIRWC ENSARLNEMGSEYYSLV TNEERRLMGT TKNNMSRTSETDNN CKKNSDSTDC KISLKAYDSK | 19 17 10 14 10 10 | Canonical sequence, contain mimic epitope (sequence shown in the underscore) control sequence, non-mimic epitope mimic epitope mimic epitope mimic epitope control sequence, non-mimic epitope |
Prepare the mimic epitope polypeptide vaccine then: listed whole mimic epitope sequences and contrast and canonical sequence in the table on the foundation, 6 of corresponding synthetic polypeptide have been made by solid-phase synthesis, be TK1, TK2, TK3 and 29A, 29B and 29C, identified the concordance of synthetic peptide sequence with mass spectral analysis.
Synthetic polypeptide is by being connected the polypeptide vaccine for preparing mimic epitope with carrier, concrete grammar is as follows: synthetic polypeptide and KLH (key hole maple hemocyanin) PBS (phosphate buffer, pH7.2) dissolving, concentration is 2mg/ml, equal-volume mixes, and dropwise adds isopyknic glutaraldehyde (0.25%), at room temperature mixing 6hr at least, the PBS that reuse contains the 50mM glycine spends the night to connecting product dialysis purification, and the gained purified product is the mimic epitope polypeptide vaccine.
Below, the effect of inducing Paralichthys olivaceus to produce cell and humoral immunoresponse(HI) of 3 kinds of mimic epitope polypeptide vaccines that the present invention is obtained detects, and experimental verification is as follows:
Paralichthys olivaceus LCDV-cn mimic epitope polypeptide vaccine to purification has carried out zoopery, to identify the specificity and the immunological effect of vaccine.The experiment picked at random is cultured totally 56 of Paralichthys olivaceuss, and body weight is about 250-300g.Be divided into 8 groups, totally 6 groups of experimental grouies, 6 kinds of polypeptide vaccines to be measured (containing reference and control vaccine, as above shown in the table) respectively are one group of 8 Paralichthys olivaceus.Contrast is established 2 groups, is divided into 4 every group, replaces polypeptide vaccine with PBS and equivalent KLH respectively and injects.
Link coupled polypeptide vaccine is with every fish dorsal intramuscular injection of concentration 200 μ l of 2mg/ml, and same dosage is injected and carried out secondary immunity after 10 days.After 26 days with 28 Paralichthys olivaceuss of 20mg/ml LCDV-cn 200 μ l/ bar counteracting toxic substances (every group of Paralichthys olivaceus got half, 4 every group of experimental grouies, 2 every group of matched groups) of purification.56, get the part Paralichthys olivaceus respectively after 76 days and slightly anaesthetize, dissect get the gill, the heart, head-kidney, spleen organize for four kinds fix with the Davidson fixative respectively or in RPMI 1640 cell culture fluids preservation standby.
The blood sampling mode is tail vein blood sampling, every fish 0.5ml/ time, and 4 ℃ of preservations are spent the night, centrifugal 10 minutes of 10000g, the collection supernatant is as the Paralichthys olivaceus antiserum.Blood sampling time (the initial immunity date is counted d0, promptly the 0th day) is respectively d-2, d3, d8, d18, d27, d38, d56, d76.
Immunological effect evaluation methodology: in order to detect the immunological effect of Paralichthys olivaceus LCDV-cn mimic epitope polypeptide vaccine; the expression that has adopted ELISA (enzyme linked immunosorbent assay analysis method) to be used for the anti-LCDV-cn IgM of Paralichthys olivaceus respectively changes and specific detection; mtt assay detects cytokine expression and lymphocytic propagation, and SABC and immunoblotting are used for the detection of viral infection situation and relative protective rate.
SABC and immunoblotting assay result show; matched group (the KLH of counteracting toxic substances; PBS) at the gill; nephridial tissue all produces positive reaction, and (one anti-is the anti-LCDV-cn multi-resistance of rabbit; tired 1: 4000); and the 29B of experimental group; 29A is negative; TK1/2/3; 29C then is shown as the weak positive; according to feminine gender to male increasing order; the result of 6 kinds of vaccines and contrast is 29B＜29A＜TK3＜TK1＜TK2＜29C＜KLH＜PBS; protective rate (meansigma methodss of four area coloring points) is followed successively by 96.4% relatively; 91.2%; 83%; 44%; 11%; 8%; 3%, 0%.The result shows; mimic epitope polypeptide vaccine of the present invention (29A, 29B and TK3) has tangible immanoprotection action; simultaneously illustrate that they are specificity vaccines at LCDV-cn, but not the control vaccine of mimic epitope sequence do not possess protective effect substantially yet.
The testing result of cytokine shows, the isolated spleen of Paralichthys olivaceus, head-kidney lymphocyte are checked through MTT behind the counteracting toxic substances, cytokine expression level and lymphocytic propagation are the highest in 29B, the A of experimental group, are respectively 0.445 ± 0.067 and 0.395 ± 0.059 and (are OD
570-OD
630Income value, down together), even be higher than the positive control (0.267 ± 0.081) that PHA stimulates, all the other are followed successively by TK3 (0.227 ± 0.051), TK1 (0.098 ± 0.043), TK2 (0.046 ± 0.038) and 29C (0.024 ± 0.030), and matched group is respectively KLH (0.040+0.037), PBS (0.018 ± 0.017).Summary is got up, and according to the order that the positive is successively decreased, its result is 29B＞29A＞PHA＞TK3＞TK1＞KLH＞29C＞PBS.The result as can be seen thus, stimulation through polypeptide, Paralichthys olivaceus lymphocyte after the immunity can be discerned antigen specifically and produce intensive cell proliferative response and improve the cytokine expression level, and lymphocyte can not be discerned control vaccine substantially as specific antigen, and its propagation is only suitable with nonspecific KLH with expression.
Following (the following OD that is of the testing result of antibody horizontal
495Optical density value), the antibody that detects in the Paralichthys olivaceus body before the immunity less than anti-LCDV-cn exists, antibody horizontal is also lower, the meansigma methods of each group is respectively 0.053 ± 0.037 (virus packets quilt, the two anti-multi-resistance that adopt the anti-Paralichthys olivaceus IgM of rabbit were tired 1: 2000) and 0.107 ± 0.087 (Paralichthys olivaceus antiserum bag quilt, total protein 50 μ g/ml, two anti-are the multi-resistance of the anti-Paralichthys olivaceus IgM of rabbit, tire 1: 2000).The 8th day the antibody horizontal in immunity back is (except that the PBS matched group: 0.062 ± 0.044) have raising, average out to 0.207 ± 0.112.But, wherein only have the antibody that produced of 29A, 29B immunity Paralichthys olivaceus have in and the LCDV-cn activity, its specific antibody expression is respectively 0.097+0.043 and 0.112 ± 0.047 (virus packets quilt), all the other experimental grouies (comprising the KLH matched group) all produce immunogenic separately antibody, each organizes average out to 0.107 ± 0.085 (experimental group polypeptide bag quilt, matched group KLH wrap quilt).
Twice peak appears in the 18th day (behind the secondary immunity 8 days) and the 38th day (behind the counteracting toxic substances 3 days), and twice antibody horizontal is respectively 0.479 ± 0.102 (experimental group+KLH matched group meansigma methods) and 0.761 ± 0.243 (counteracting toxic substances cell mean).Wherein, have only 29A and 29B to produce specific antibody, be respectively 0.385 ± 0.079/0.789 ± 0.153 and 0.482 ± 0.094/0.906 ± 0.183 (the 18/38th day antibody test value of 29A, 29B).After this, the antibody horizontal of 29A, 29B counteracting toxic substances group sharply descends, reduce to 0.142 ± 0.056 to d76, all the other each groups then maintain more stable level substantially, be about 0.351 ± 0.120, and in the counteracting toxic substances group (except that 29A, 29B), all can detect the antibody of the anti-LCDV-cn of Paralichthys olivaceus at d56, d76, its average is 0.122 ± 0.063.
Above-mentioned antibody test result shows that mimic epitope polypeptide vaccine 29A and 29B can induce propagation, the differentiation of B cell, produce specific humoral immunoresponse(HI), and cause the immunological memory effect, thereby effectively improves the immunoprophylaxis ability of Paralichthys olivaceus to LCDV-cn.
Therefore; the immunological effect testing result of 3 kinds of mimic epitope polypeptide vaccines that the present invention makes presents the unanimity of height; the mimic epitope that the bioinformatics design that the present invention adopts is described has strong immunogenicity and immunoreactivity; and the polypeptide vaccine that constructs in view of the above can induce Paralichthys olivaceus to produce specific body fluid and cellullar immunologic response, and higher relative protective rate also can be provided simultaneously.In addition, the comparing result of mimic epitope polypeptide vaccine and control vaccine immunological effect proves that also mimic epitope of the present invention is consistent with the antigenic determinant of LCDV-cn protective antigen nucleocapsid protein and envelope protein.Based on above-mentioned, the three kinds of mimic epitope polypeptide vaccines (29A, 29B and TK3) among the present invention can play effective immunoprophylaxis and protective effect.
This method prepares easy.Different with the incomplete immunization of inactivated vaccine, the mimic epitope polypeptide vaccine can effectively induce body to produce comprehensive cell and humoral immunoresponse(HI); And compare with attenuated vaccine, polypeptide vaccine has very high safety again, therefore, and the succedaneum that the vaccine of this method preparation can produce as the advantage that combines traditional attenuation, inactivated vaccine.
Claims (4)
1, the preparation method of the polypeptide vaccine of lefteye flonder lymphocystic disease, the bioinformatic analysis that known lymphocystis disease virus Europe separated strain genome sequence is carried out and obtain 3 mimic epitopes of lefteye flonder lymphocystic virus Chinese pathogenic strain nucleocapsid protein and envelope protein at first, then according to the aminoacid sequence of mimic epitope, synthesize and obtain corresponding 3 peptide species, through with the coupling and the purification of protein carrier, promptly prepared the mimic epitope polypeptide vaccine of lefteye flonder lymphocystic disease.
2, the preparation method of the polypeptide vaccine of lefteye flonder lymphocystic disease as claimed in claim 1 is characterized in that 3 above-mentioned mimic epitope sequences are respectively:
29A：NH
2-Thr-Lys-Asn-Asn-Met-Ser-Arg-Thr-Ser-Glu-Thr-Asp-Asn-Asn-COOH
29B：NH
2-Cys-Lys-Lys-Asn-Ser-Asp-Ser-Thr-Asp-Cys-COOH
TK3：NH
2-Thr-Asn-Glu-Glu-Arg-Arg-Leu-Met-Gly-Thr-COOH。
3, the preparation method of the polypeptide vaccine of lefteye flonder lymphocystic disease as claimed in claim 1 is characterized in that described protein carrier is a key hole maple hemocyanin.
4, the preparation method of the polypeptide vaccine of lefteye flonder lymphocystic disease as claimed in claim 1 is characterized in that described protein carrier is hemoglobin, bSA.
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| WO2011124056A1 (en) * | 2010-04-06 | 2011-10-13 | 国家海洋局第一海洋研究所 | Genetically engineered vaccine against lymphocystis disease in paralichthys olivaceus and method for producing the same |
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| CN1322008C (en) * | 2004-12-17 | 2007-06-20 | 中国海洋大学 | Monoclonal antibody of anti lymphocyst virus of and preparation method |
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| WO2011124056A1 (en) * | 2010-04-06 | 2011-10-13 | 国家海洋局第一海洋研究所 | Genetically engineered vaccine against lymphocystis disease in paralichthys olivaceus and method for producing the same |
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